Influence of pH on Enzymatic Extract with Allelopathic Potential

PETRU NEGREA1, ADINA NEGREA1, MARILENA MOTOC2, MIHAELA CIOPEC1, ANDREEA GABOR1, CORNELIA MUNTEAN1*
1
University Politehnica Timisoara, Faculty of Industrial Chemistry and Environmental Engineering, 2 Victoriei Sq., 300006,
Timisoara, Romania
2
Victor Babes University of Medicine and Pharmacy Timisoara, 2 Eftimie Murgu Sq., 300041, Timisoara, Romania

Redox processes that involve enzymatic extract with allelopathic potential are accompanied by changes in
the pH value of the environment. In this paper was shown using UV-Vis spectrometry that the allelopathic
potential depends on the external environment. To determine the influence of the pH of the environment on
electrochemical behavior of nitrophenols we worked in buffered medium. pH value was adjusted using 0.1
M NaOH solution. This study showed that the enzyme extract with allelopathic potential in the state of
oxidation, and in the protonated form, has a low oxidation potential, which shows that it is a typical reductant.
The results indicated that the change in pH affects the mode of action of allelopathic extracts. Based on
these considerations it can be concluded that the UV-VIS spectrophotometry is a suitable method to quickly
determine the concentration of polyphenols from extracts with allelopathic potential extracts from
transformation-adsorption processes and the natural processes of soil polyphenol derivatives.
Keywords: enzymatic extract, allelopathic potential, pH environment, polyphenolic derivatives

Allelopathy is a natural organic technique, which proved

useful as an unique tool for weed management. Soil is a
heterogeneous system, multiphase, dispersed, divided,
porous, dynamic, and animated. Heterogeneity is
conditioned by variation in the characteristics of the soil
mass as a whole or in its parts [1]. Soil is a multiphase
system having a mixed composition: solid phase, liquid
and gas. The solid phase, named matrix of the soil, is
composed by mineral constituents, coming from the
transformation of the parent material, and organic
constituents, resulting in the transformation of the material
incorporated into the soil [2].
Redox processes from the soil are conditioned by the
activity of microorganisms [3], which using the energy
resulting from the decomposition of living organisms,
perform the oxidation by electron transfer from organic
substances (reducing agents) to other substances which
act as electron acceptor (oxidizing agents) [4].
Oxygen is in this particular case one of the most powerful
electron acceptor, resulting in the release of a large amount
of energy in the oxidation process of organic matter.
Oxygen represents the only acceptor of electrons that
plants can use [5]. In order to have optimal growth and
development of plants, it is necessary that in the soil,
oxidation processes predominate over reduction
processes. Hydrogen participates in oxidation-reduction
reactions, and the value of the redox potential will be
affected by the pH of the medium (the value of the redox
potential increases with decreasing pH) [6]. The oxidationreduction conditions in the soil influence the mobility of
macro- and micro-nutrients [7]. In total anaerobic
conditions (water stagnation on the ground), redox potential
is negative, causing the complete reduction of metals and
sulphates to elemental sulfur and sulphide (H2S-toxic to
plants) [8]. The activity of aerobic microorganisms is
blocked, occurring anaerobic fermentation with the
formation of methane and other gases that are toxic to
plants [9].

Oxidability of the soil represents the negative logarithm

of the activity of free electrons. Oxidability of the soil varies
between -6 and +13; high values of the oxidability indicate
that the chemical species are in the oxidized state, and
low values indicate that chemical species are in the
reduced state [10]. Aerobic microorganisms have not
activity when the oxidability values are <5; denitrifying
bacteria act in the range +10 and 0; bacteria that reduce
sulfates live when the values are under 2.
Sorghum halepense contains large amounts of phenolic
compounds with allelopathic potential (p-hydroxybenzoic
acid, p -coumaric acid, and trans-cinnamic acid) and
dhurrin, a cyanogenic glycoside that releases hydrogen
cyanide after hydrolysis [11]. The amount of dhurrin
depends on growing conditions, fertilization, temperature
and water availability [12]. To prevent contamination of
the land after frost or surges of water, it should be avoided,
and it is recommended to dry the plants, as HCN resulting
from hydrolyzed dhurrin is volatile.
An important role is played by the soil pH value [13]. In
order that the easily oxidable extract to be useful, is
recommended a relatively neutral pH of the environment.
By increasing the pH value from 0 to 6, the oxidation of the
potential allelopathic enzyme extract occurs generally
easily, while its reduction (by adding soil solution and
leachate) takes place more difficult. At each pH value, the
system reaches the equilibrium corresponding to a defined
oxidation state of the enzymatic extract with allelopathic
potential [14]. As the p H of the medium and the
concentration of the oxidant are higher, the oxidation state
of the enzymatic extract with allelopathic potential is
higher.
Thus, if the system does not contain a buffer, it must be
taken into account both the initial p H and the p H
corresponding to the state of equilibrium [15]. Increasing
the pH to values above 6, excludes the possibility of
reducing the extract with allelopathic potential by the
addition of soil solution and leachate. For this reason, the
enzymatic extract with allelopathic potential will be

* email: cornelia.muntean@upt.ro, Tel.: +40 256 404192

372

http://www.revistadechimie.ro

REV.CHIM.(Bucharest) 67 No.2 2016

Experimental part
Materials and methods
Processing of the samples to be analyzed: Portions of
10 g from the vegetal material (Sorghum halepense) finely
pulverized were extracted at room temperature by
maceration with 100 mL methanol for 24 h. There were
analyzed two samples in parallel, one as such and the other
hydrolyzed. Due to the presence of at least one phenolic
function in the molecule (but also the presence of a
carboxyl group in the case of polyphenolic acids), all of the
compounds can lose an acid proton (turning into negative
ions). Caffeic acid, chlorogenic acid, ferulic acid, pcoumaric acid and polyphenol acids are most commonly
encountered in the analyzed species. The electronic
absorption spectra of the dispersed enzyme extract of
Sorghum halepense at different pH values were recorded
with an UVVIS spectrophotometer (T60U, PG Instruments
Limited, used in connection with the associated software
WIN UV version 5.05) using quartz cuvettes of 1 cm
thickness [17].
Results and discussions
From the UV-Vis molecular absorption spectra recorded
for polyphenolic compounds found in soil solution and
leachate, shown in figure 1, one can notice that the
absorption peaks are well separated. Every UV-Vis
spectrum shows at least two absorption bands with
different values of wavelength of maximum absorbance.
Although the calibration data have been derived for two
values of max, for each compound [18], only one of them
was used for the study due to high values of the absorption
coefficients ( = 9330 at 400 nm, = 5970 at 440 nm,
= 6280 at 390 nm, and = 5450 at 470 nm, respectively).
All calibration curves recorded for the polyphenolic
compounds studied pass through the origin and show good
correlation coefficients, which allows the estimation of
concentration of these compounds solutions by means of
spectrophotometric measurements and equations that
describe the dependence of absorbance on concentration
[19].
The optimal concentration range where one may use
UV-VIS spectrophotometry to determine the polyphenolic
compounds studied, in aqueous medium, is: 0.220 mg
L-1 (limit concentration accepted: 0.5 mg L-1).
When the enzyme extract with allelopathic potential
(as an aqueous dispersion) was treated using solutions
with pH values between 0 and 11, were registered the
spectra of the compounds which absorb in the blue zone
(fig. 2), that is, those corresponding to the oxidation states.
At each pH value, the oxidation of the enzyme extract
with allelopathic potential may be carried out to the highest
degree of oxidation, if during the increase of pH is added
an oxidant which amount decreases normally [20]. The
electrochemical potential of the system, corresponding to
the formation of stable extract, decreases at higher pH. By
acidification to pH < 6, the blue mixture and the mixture
of compounds formed in neutral and alkaline media is
capable of reduction, by addition of soil solution and
leachate, until the equilibrium of the oxidation state is
reached [21]. The oxidation of enzyme extract in the acidic
REV.CHIM.(Bucharest) 67 No.2 2016

(a. u.)

oxidized to various oxidation states of equilibrium, which

depend on the oxidation potential and on the concentration
of the oxidant present in the system (residual oxidant and
oxygen) [16]. If the oxidation potential of these oxidizing
agents does not depend on pH, the equilibrium of oxidation
state of the extract will be unchanged over the entire pH
range corresponding to neutral and alkaline environment.

Fig. 1. UV-VIis spectra of polyphenol derivatives

Fig. 2. Electronic absorption spectra of the dispersed enzyme

extract with allelopathic potential, at different pH values

environment, in the potential range where soil solution and

leachate serving as internal protector where already
exhausted, led to formation of the same organic blue
products. As expected, neutralizing these oxidized forms
of the enzyme extract with allelopathic potential obtained
in neutral and alkaline media and then subjected to
acidification, does not lead to changes in the oxidation
state and in the electronic absorption spectra of these
extracts.
The explanation could be made based on the alternative
concept of transition allelopathic compound salt allelopathic compound base [22]. Under this approach,
the enzyme extract in state of active compound is oxidized
when is treated under neutral or alkaline (pH > 6) medium.
The resulting base compound (blue product characterized
by max in the visible at 420 nm) represents the enzyme
extract with allelopathic potential in an intermediate
oxidation state, between the active compound and soil
solution and leachate [23]. The changes in the structure of
the enzyme extract with allelopathic potential arising by
treatment of the compound in environments with different
pH affects the electronic absorption spectrum [24]. This
can be explained by the degree of protonation of the imine
nitrogen atoms in the enzyme extract found in the oxidation
state of active compound [25].
The highest degree of protonation and the highest
electrical conductivity were achieved at a pH between 0
and 1, when the salt form of the active compound was
obtained. The deprotonated form (or the base active
compound), which seems to be insulating, was obtained
in a neutral or alkaline medium [26]. The transition active
salt compound - active base compound was named nonoxidative doping [27]. The activity of the enzyme extract
depends on two variables, namely, the oxidation degree
and the protonation degree. This means that one can
distinguish two types of doping, the oxidative and the nonoxidative, respectively. Optical analyzes on the enzyme
extract with allelopathic potential, carried out to track the
kinetics of color change in electrochromic experiments
led to some fundamental speculations. Their main point is

http://www.revistadechimie.ro

373

that oxidation of some bonds in the active base compound

necessarily passes through the intermediate state active
salt compound, which corresponds to the claim that the
allelopathic base compound is characterized by the highest
state of oxidation [28].
The processes of protonationdeprotonation of the
enzyme extract with allelopathic potential showed that
during the deprotonation takes place an increase of the
oxidation state, while a subsequent treatment with an acid,
causes a decrease in this state.
Conclusions
The oxidizing-reducing behaviour of the compounds
contained in the enzyme extract with allelopathic potential
cannot be separated from environmental pH. The influence
of the pH of the medium on the properties of the compounds
found in the enzyme extract cannot be examined without
taking into account the redox processes occurring in the
case of this extract.
The pH value of the medium appears to be one of the
most important parameters, which significantly affects the
direction of redox processes involving the compounds of
the enzyme extract with allelopathic potential. Thus, the
pH value determines the oxidation state of the compounds,
which in turn determines the properties of the extract.
In order to choose the best method, should be considered
the possibility that the oxidation state changes during the
analysis, which is impossible without expertise in
electrochemical activity of the enzyme extract with
allelopathic potential.
There is no consensus on investigations carried out by
elemental analysis, as it gives only sketchy indications on
the chemical structure. Spectroscopic techniques such as
IR spectroscopy and electronic absorption spectroscopy
only give qualitative indication of the oxidation state of the
compounds of the enzyme extract with allelopathic
potential. Even the X-ray photoelectron spectroscopy is an
analysis that does not allow for accurate resolution of the
peaks corresponding to imine and amine nitrogen atoms.
A suitable quantitative technique would be titration of
the compounds of the enzyme extract but under the
conditions used could be many errors.
It can be concluded that the UV-Vis spectrophotometry
is a suitable method to quickly determine the concentration
of polyphenols from extracts with allelopathic potential
extracts. The importance of precise determination of the
oxidation state of the enzyme extract with allelopathic
potential is related to the effects of this mixture of
substances on the sustainability of the environment.

3.BUTU, M. RODINO, S. BUTU, A. BUTNARIU, M. Dig. J. Nanomater.

Bios., 9, no. 2, 2014, p. 519.
4.BUTNARIU, M. CAUNII, A. Ann. Agric. Environ. Med., 20, no. 4, 2013,
p. 736.
5.FERENCZ, A; JUHASZ, R; BUTNARIU, M; DEER, AK; VARGA, IS;
NEMCSOK, J. Acta. Biol. Hung., 63, no. 1, 2012, 15.
6.BUTNARIU, M; BOSTAN, C. African journal of biotechnology., 10,
no. 31, 2011, p. 5900.
7.CAUNII, A. NEGREA, A. PENTEA, M. SAMFIRA, I. MOTOC, M.
BUTNARIU, M. Rev. Chim. (Bucharest), 66, no. 3, 2015, p. 382.
8.IANCULOV, I. GERGEN, I. PALICICA, R. BUTNARIU, M. DUMBRAVA,
D. GABOR, L. Rev. Chim. (Bucharest), 55, no. 11, 2004, p. 835.
9.BUTNARIU, M. RODINO, S. PETRACHE, P. NEGOESCU, C. BUTU, M.
Dig. J. Nanomater. Bios., 9, no. 2, 2014, p. 745.
10.CAUNII, A; BUTU, M; RODINO, S; MOTOC, M; NEGREA, A; SAMFIRA,
I; BUTNARIU, M, Rev. Chim. (Bucharest), 66, no. 4, 2015, p. 472.
11.BOSTAN, C. BUTNARIU, M. BUTU, M. ORTAN, A. BUTU, A. RODINO,
S. PARVU, C. Rom. Biotech. Lett., 18, no. 2, 2013, p. 8190.
12.BUTNARIU, M; CAUNII, A; PUTNOKY, S. Chem. Cent. J. 6, 2012, 146.
13.BARBAT, C. RODINO, S. PETRACHE, P. BUTU, M. BUTNARIU, M.
Dig. J. Nanomater. Bios., 8, no. 3, 2013, p. 945.
14.BUTNARIU, M. SMULEAC, A. DEHELEAN, C, CHIRITA, R. SARATEAN,
V. Rev. Chim. (Bucharest), 57, no. 11, 2006, p. 1138.
15.PUTNOKY, S; CAUNII, A; BUTNARIU, M. Chem. Cent. J., 7, 2013,
146.
16.BUTNARIU, M. GOIAN, M. IANCULOV, I. GERGEN, I. NEGREA, P.
Rev. Chim. (Bucharest), 56, no. 8, 2005, p. 837.
17.BUTNARIU, M. CORADINI, C. Z. Chem. Cent. J, 6, 2012, 35.
18.BUTU, M. RODINO, S. PENTEA, M. NEGREA, A. PETRACHE, P.
BUTNARIU, M. Dig. J. Nanomater. Bios., 9, no. 4, 2014, p. 1317.
19.RODINO, S. BUTU, M. NEGOESCU, C. CAUNII, A. CRISTINA, R. T.
BUTNARIU, M. Dig. J. Nanomater. Bios., 9, no. 3, 2014, p. 1215.
20.BUTU, M. BUTNARIU, M. RODINO, S. BUTU, A. Dig. J. Nanomater.
Bios., 9, no. 3, 2014, p. 935.
21.PENTEA, M. BUTU, M. SAMFIRA, I. CRISTINA, R. T. BUTNARIU, M.
Dig. J. Nanomater. Bios., 10, no. 1, 2015, p. 291.
22.PETRACHE, P. RODINO, S. BUTU, M. PRIBAC, G. PENTEA, M.
BUTNARIU, M. Dig. J. Nanomater. Bios., 9, no. 4, 2014, p. 1523.
23.BUTU, M. BUTNARIU, M. RODINO, S. BUTU, A. Dig. J. Nanomater.
Bios., 9, no. 3, 2014, p. 935.
24.RASHED, K. BUTNARIU, M. Iran. J. Pharm. Res., 13, no. 3, p. 2014,
1073.
25.BUTU, M. RODINO, S. BUTU, A. BUTNARIU, M. Dig. J. Nanomater.
Bios., 9, no. 2, 2014, p. 519.
26.CIOPEC, M; NEGREA, A; PENTEA, M; SAMFIRA, I; MOTOC, M;
BUTNARIU, M. Rev. Chim. (Bucharest), 66, no. 5, 2015, p. 645.
27.BUTNARIU, M. V.; GIUCHICI, C. V. Journal of nanobiotechnology, 9,
2011, 3.
28.BAGIU, R.V. VLAICU, B. BUTNARIU, M. Int. J. Mol. Sci., 13, no. 2,
2012, p. 1426

References
1.BUTNARIU, M. Chem. Cent. J., 6, 2012, 75.
2.IANCULOV, I. PALICICA, R. BUTNARIU, M. DUMBRAVA, D. GERGEN,
I. Rev. Chim. (Bucharest). 56, no. 4, 2005, p. 441.

374

Manuscript received: 18.08.2015

http://www.revistadechimie.ro

REV.CHIM.(Bucharest) 67 No.2 2016